Antioxidant defense against antidepressants in C6 and 1321N1 cells
Introduction
The mechanisms underlying the actions of antidepressants are not clearly understood. The latency of clinical response may not be attributable to their acute pharmacological actions and considerable evidence suggests that the signalling pathways which regulate gene transcription through the cAMP and Ca2+ response element may be affected at multiple levels (for review see [1]). The downregulation of 5HT2 and β-adrenergic receptors and blocking of voltage-dependent calcium channels, together with alterations in G-protein activities are well documented changes [2], [3]. However, most antidepressants also possess significant toxicities and the incidence of overdosing is high [4]. Despite the widely documented neurological impairments, which may be caused by antidepressants, there are no available data on the underlying processes.
A considerable amount of information on antidepressants has been obtained from in vitro studies. These include pharmacological, biochemical and toxicological studies on both neurons and astrocytes. The astrocytes in particular have attracted attention because both short and relatively long-term culture studies can be made which may more closely reflect the effects of acute or chronic treatments. For example, some of the earlier studies demonstrated that chronic exposure of primary mouse astrocytes to antidepressants downregulated adenylyl cyclase linked β-adrenergic receptors with a time course which paralleled that seen in vivo in animal brain homogenates and the therapeutic improvement seen in depressed patients [5]. The great potential importance of astrocytes in the therapeutic effects of antidepressants and other neuroactive drugs was thus established [6].
There is now substantial evidence that the astrocytes have key protective capacities against a range of potentially damaging effects in the CNS and that these capacities may be increased in reactive states [7], [8]. Among these, the antioxidant defence system appears especially effective and becomes accentuated following reactive gliosis [8], [9].
In a recent study, we analysed the effects of several antidepressants in cultured rat C6 glioma and human (132#1N1) astrocytoma cell lines using a number of astrocyte-specific, metabolic and cytotoxic methods [10]. This work demonstrated that there was an overall good correlation between EC50 values in the cell lines and LD50 values (RTECS listed) for the antidepressants tested, with several substances categorised as extremely toxic. However, it also showed that the antidepressants could initiate activation-type responses at subcytotoxic doses. These were evident as increases in GFAP levels with increased branching of cell processes and increases in MTT conversion and neutral red uptake. Other workers have previously shown that treatment of astrocyte cultures with β-adrenergic agonists or dibutyryl cyclic AMP (dBcAMP) initiate biochemical and morphological changes which have been used to model aspects of reactive astrocytes in vitro [7], [11], [12], [13], [14]. We have also found that these changes are associated with increased protective capacities against a number of toxic substances [15].
The purpose of the present work was to assess the protective capacities of astrocytes against the acute effects of antidepressants using two astrocytes cell lines. Cells were exposed to antidepressants and antioxidant levels manipulated by drug treatments. In some experiments, the levels of cyclic AMP (cAMP) were altered to induce activation-type responses. The work shows that the cells’ defence systems protected against damage caused by the antidepressants and that the damage might be caused by oxidative stress. Modifications of the cAMP levels indicated that activation-type responses might confer increased protective capacity.
Section snippets
Materials
All materials were obtained from Sigma unless otherwise stated. C6 and 1321N1 cells were obtained from the European collection of animal cell cultures at Porton down, Salisbury, UK.
Cell culture methods
C6 and 1321N1 cells were subcultured from stock flasks according to our previous descriptions [10]. In brief, cells were washed in Hanks balanced salt solution (HBSS, 3×5 ml), trypsinised (1 ml trypsin-EDTA), and diluted with 9 ml of astrocyte growth medium (AGM). The AGM used was DMEM supplemented with 10% (v/v)
Effects of antioxidants
The EC50 value changes in C6 and 1321N1 cells after antioxidant treatment are summarised in Table 1, Table 2. There were significant protective shifts after GSH treatment in C6 cells (pretreated with dBcAMP), this was greatest for fluoxetine (Table 1). Buthionine-[S,R]-sulfoximine (BSO) which depletes intracellular glutathione levels enhanced the toxicity of some of the antidepressants (negative shifts in EC50 value) in C6 cells (+dBcAMP), although these changes were not significant. In
Discussion
The antidepressants were chosen for study because they represent the major classes of antidepressants (i.e. the monoamine oxidase inhibitors, tricyclics and selective serotonin re-uptake inhibitors) and are also the widely prescribed antidepressants of the classes. In contrast to the extensive literature on the pharmacological and biochemical processes which may underlie the beneficial therapeutic effects of the antidepressants, very little is known about the ways they may initiate changes in
Acknowledgements
This work was funded by the Science Research Institute (Salford University). The authors would like to thank Dr G. Hide for statistical assistance.
References (32)
- et al.
The effects of antidepressant drugs on adenylyl cyclase linked beta adrenergic binding sites on mouse astrocytes in primary cultures
Prog. Neuro-Psychopharmacol. Biol. Psychiatry
(1983) - et al.
Is neuropharmacology merely the pharmacology of neurons or are astrocytes important too?
TIPS Rev.
(1984) - et al.
Molecular profile of reactive astrocytes — implications for their role in neurologic disease
Neuroscience
(1993) - et al.
Interrelationships between astrocyte function, oxidative stress and antioxidant status within the central nervous system
Prog. Neurobiol.
(1997) - et al.
Enzymatic and morphological properties of primary rat brain astrocyte cultures and enzyme development in vivo
Brain Res.
(1978) - et al.
Organization of microfilaments in astrocytes that form in the presence of dibutyryl cyclic AMP in cultures, and which are similar to reactive astrocytes in vivo
Neuroscience
(1987) - et al.
Responses in astrocytic C6 glioma cells to ethanol and dibutyryl cyclic AMP
Dev. Brain Res.
(1986) - et al.
Hypertrophy and increased glial fibrillary acidic protein are coupled to increased protection against cytotoxicity in glioma cell lines
Toxicol. In Vitro
(1998) Determination of glutathione and glutathione disulfide in biological samples
Methods Enzymol.
(1985)- et al.
Protective roles of glutathione in the toxicity of mercury and cadmium compounds to C6 glioma cells
Toxicol. In Vitro
(1996)
Myocardial salvage with Trolox and ascorbic acid for an acute evolving infarction
Ann. Thorac. Surg.
Receptors on astrocytes — what possible functions?
Neurochem. Int.
Pharmacokinetic and pharmacodynamic significance of antidepressant drug metabolites
Pharmacol. Res.
Fluoxetine tenth anniversary update: the progress continues
Clin. Therapeutics
Progress in defining the mechanism of action of antidepressants. Across receptors and into gene transcription
CNS Drugs
Effects of long-term administration of antidepressants and neuroleptics on receptors in the central nervous system
Cell. Mol. Neurobiol.
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Present address: Development DMPK II Alderley, AstraZeneca Pharmaceuticals, Alderley Park, Alderley Edge, Macclesfield SK1O 4TG, UK.